Gene involved in apoptosis regulation

ABSTRACT

This invention encompasses sequences of nucleic acids and polypeptides that are involved in the regulation of tumor suppression and apoptosis and methods of using the sequences. Methods of identification for compounds that are effective in treating disorders such as cancer and neurodegenerative disease are also disclosed.

The present invention relates to a nucleic acid sequence involved in theregulation of apoptosis and of tumor reversion by the p53 pathway, andalso to the protein encoded by this nucleic acid sequence.

Apoptosis, or cell death, is a complex phenomenon which is regulated bymany proteins, including the p53 protein. This protein interacts withmany other proteins, and its expression, which induces the phenomena ofcell death and of tumor reversion, can be correlated with the inductionor the suppression of the expression of other cellular genes.

The inventors of the present invention have thus demonstrated geneswhich are induced and activated during the cascade leading to tumorsuppression and/or apoptosis (TSAP for “Tumor Suppressor ActivatedPathway”), or genes which are suppressed (TSIP for “Tumor SuppressorInhibited Pathway”). These genes were in particular the subject ofpatent applications WO 97/22695 and WO 00/08147.

It is important to be able to understand precisely the mechanisms of thep53 cascade, in order to be able to generate novel compounds havingantitumor activity (which can in particular induce apoptosis or tumorsuppression), or which can be used for the treatment ofneurodegenerative diseases. In fact, the inventors of the presentinvention have demonstrated that presenilin 1 (PS1), the role of whichhad been suggested in Alzheimer's disease, is identical to the TSIP 2protein described in application WO 97/22695. Thus, it is legitimate tosearch for medicinal products which can interfere in apoptosis, in orderto reduce this phenomenon, and which might be used in neurodegenerativediseases.

The inventors of the present application have demonstrated theinteraction of the TSIP 2 protein (presenilin 1, PS1) with a proteinwhich has itself been shown to be repressed in a model of cell death.This protein (SEQ ID No. 2) was thus called TSIP4, and it is a subjectof the present invention, as are the nucleic acid sequences which encodesaid protein. The TSIP4 gene will in particular be preferred, it beingunderstood that the term “gene” may be intended to mean either the cDNAsequence or the genomic DNA sequence, with or without the regulatoryelements.

Thus, a subject of the present invention is a purified or isolatednucleic acid, characterized in that it comprises a nucleic acid sequencechosen from the group of following sequences:

-   -   a) SEQ ID No. 1;    -   b) the sequence of a fragment of at least 15 consecutive        nucleotides of a sequence chosen from SEQ ID No. 1;    -   c) a nucleic acid sequence exhibiting a percentage identity of        at least 80%, after optimal alignment, with a sequence defined        in a) or b);    -   d) a nucleic acid sequence which hybridizes, under high        stringency conditions, with a nucleic acid sequence defined        in a) or b);    -   e) the complementary sequence or the RNA sequence corresponding        to a sequence as defined in a), b), c) or d).

The nucleic acid sequence according to the invention defined in c)exhibits a percentage identity of at least 80%, after optimal alignment,with a sequence as defined in a) or b) above, preferably 90%, morepreferably 95%, most preferably 98%, or 99%.

The terms “nucleic acid”, “nucleic acid sequence”, “polynucleotide”,“oligonucleotide”, “polynucleotide sequence” and “nucleotide sequence”,terms which will be used indifferently in the present description, areintended to denote a precise series of nucleotides, which may or may notbe modified, making it possible to define a fragment or region of anucleic acid, possibly comprising unnatural nucleotides, and which maycorrespond equally to a double-stranded DNA, a single-stranded DNA andproducts of transcription of said DNAs. Thus, the nucleic acid sequencesaccording to the invention also encompass PNAs (peptide nucleic acids),or the like.

It should be understood that the present invention does not concern thenucleotide sequences in their natural chromosomal environment, i.e. inthe natural state. They are sequences which have been isolated and/orpurified, i.e. they have been removed directly or indirectly, forexample by copying, their environment having been at least partiallymodified. The nucleic acids obtained by chemical synthesis are thus alsointended to be denoted.

For the purpose of the present invention, the term “percentage identity”between two nucleic acids or amino acid sequences is intended to denotea percentage of nucleotides or of amino acid residues which areidentical between the two sequences to be compared, obtained after thebest alignment, this percentage being purely statistical and thedifferences between the two sequences being distributed randomly andover their entire length. The term “best alignment” or “optimalalignment” is intended to denote the alignment for which the percentageidentity determined as below is highest. The sequence comparisonsbetween two nucleic acid or amino acid sequences are conventionallycarried out by comparing these sequences after having optimally alignedthem, said comparison being carried out by segment or by “window ofcomparison” so as to identify and compare local regions of sequencesimilarity. The optimal alignment of the sequences for the comparisoncan be carried out, besides manually, by means of the local homologyalgorithm of Smith and Waterman (1981), by means of the local homologyalgorithm of Neddleman and Wunsch (1970), by means of the similaritysearch method of Pearson and Lipman (1988), by means of computerprograms using these algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTAand TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Drive, Madison, Wis.). In order to obtain the optimalalignment, the BLAST program is preferably used, with the BLOSUM 62matrix. The PAM or PAM250 matrices can also be used.

The percentage identity between two nucleic acid or amino acid sequencesis determined by comparing these two optimally aligned sequences, thenucleic acid or amino acid sequence to be compared possibly comprisingadditions or deletions compared to the reference sequence for optimalalignment between these two sequences. The percentage identity iscalculated by determining the number of identical positions for whichthe nucleotide or the amino acid residue is identical between the twosequences, dividing this number of identical positions by the totalnumber of positions compared, and multiplying the result obtained by 100so as to obtain the percentage identity between these two sequences.

The expression “nucleic acid sequences exhibiting percentage identity ofat least 80%, preferably 90%, more preferably 98%, after optimalalignment with a reference sequence” is intended to denote the nucleicacid sequences which, compared to the reference nucleic acid sequence,exhibit certain modifications, such as in particular a deletion, atruncation, an extension, a chimeric fusion and/or a substitution, inparticular of the point type, and the nucleic acid sequence of whichexhibits at least 80%, preferably 90%, more preferably 98%, identity,after optimal alignment, with the reference nucleic acid sequence. Theyare preferably sequences whose complementary sequences are capable ofhybridizing specifically with the sequences SEQ ID No. 1 of theinvention. Preferably, the specific or high stringency hybridizationconditions will be such that they ensure at least 80%, preferably 90%,more preferably 98%, identity, after optimal alignment, between one ofthe two sequences and the sequence complementary to the other.

Hybridization under high stringency conditions means that the conditionsof temperature and of ionic strength are chosen such that they allow thehybridization between two complementary DNA fragments to be maintained.By way of illustration, high stringency conditions for the hybridizationstep for the purposes of defining the polynucleotide fragments describedabove are advantageously as follows.

The DNA-DNA or DNA-RNA hybridization is carried out in two steps: (1)prehybridization at 42° C. for 3 hours in phosphate buffer (20 mM, pH7.5) containing 5×SSC (1×SSC corresponds to a solution of 0.15 MNaCl+0.015 M sodium citrate), 50% of formamide, 7% of sodium dodecylsulfate (SDS), 10× Denhardt's, 5% of dextran sulfate and 1% of salmonsperm DNA; (2) hybridization per se for 20 hours at a temperature whichdepends on the length of the probe (i.e.: 42° C. for a probe>100nucleotides in length), followed by two washes of 20 minutes at 20° C.in 2×SSC+2% SDS, and 1 wash of 20 minutes at 20° C. in 0.1×SSC+0.1% SDS.The final wash is carried out in 0.1×SSC+0.1% SDS for 30 minutes at 60°C. for a probe>100 nucleotides in length. The high stringencyhybridization conditions described above for a polynucleotide of definedlength can be adjusted by those skilled in the art for longer or shorteroligonucleotides, according to the teaching of Sambrook et al., 1989.

Among the nucleic acid sequences exhibiting a percentage identity of atleast 80%, preferably 90%, more preferably 98%, after optimal alignment,with the sequence according to the invention, preference is also givento the nucleic acid sequences which are variants of SEQ ID No. 1, or ofits fragments, i.e. all of the nucleic acid sequences corresponding toallelic variants, i.e. individual variations of the sequence SEQ IDNo. 1. These natural mutated sequences correspond to polymorphismspresent in mammals, in particular in humans, and in particular topolymorphisms which may lead to the occurrence of a pathologicalcondition, such as, for example, a cell degeneration. Preferably, thepresent invention relates to the variant nucleic acid sequences in whichthe mutations lead to a modification of the amino acid sequence of thepolypeptide, or of its fragments, encoded by the normal sequence of SEQID No. 1.

The expression “variant nucleic acid sequence” is also intended todenote any RNA or cDNA resulting from a mutation and/or variation of asplice site of the genomic nucleic acid sequence, the cDNA of which hasthe sequence SEQ ID No. 1.

The invention preferably relates to a purified or isolated nucleic acidaccording to the present invention, characterized in that it comprisesor consists of the sequence SEQ ID No. 1, the sequence complementarythereto, or the RNA sequence corresponding to SEQ ID No. 1.

The invention also relates to a purified or isolated nucleic acid,characterized in that it encodes a polypeptide having a continuousfragment of at least 100, more preferably 150, most preferably 200,amino acids of the protein SEQ ID No. 2.

The primers or probes, characterized in that they comprise a sequence ofa nucleic acid according to the invention, are also part of theinvention.

Thus, the present invention also relates to the primers or the probesaccording to the invention which may make it possible, in particular, todemonstrate or to discriminate the variant nucleic acid sequences, or toidentify the genomic sequence of the genes the cDNA of which isrepresented by SEQ ID No. 1, using in particular an amplification methodsuch as the PCR method, or a related method.

The invention also relates to the use of a nucleic acid sequenceaccording to the invention as a probe or primer, for detecting,identifying, assaying or amplifying a nucleic acid sequence.

According to the invention, the polynucleotides which can be used as aprobe or as a primer in methods for detecting, identifying, assaying oramplifying nucleic acid sequence are a minimum of 15 bases, preferablyof 20 bases, or better still of 25 to 30 bases, in length.

The probes and primers according to the invention can be directly orindirectly labeled with a radioactive or nonradioactive compound usingmethods well known to those skilled in the art, in order to obtain adetectable and/or quantifiable signal.

The polynucleotide sequences according to the invention which areunlabeled can be used directly as a probe or primer.

The sequences are generally labeled in order to obtain sequences whichcan be used for many applications. The labeling of the primers or of theprobes according to the invention is carried out with radioactiveelements or with nonradioactive molecules.

Among the radioactive isotopes used, mention may be made of ³²P, 33P,³⁵S, ³H or ¹²⁵I. The nonradioactive entities are selected from ligandssuch as biotin, avidin, streptavidin or digoxigenin, haptens, dyes, andluminescent agents such as radioluminescent, chemiluminescent,bioluminescent, fluorescent or phosphorescent agents.

The polynucleotides according to the invention can thus be used as aprimer and/or probe in methods using in particular the PCR (polymerasechain reaction) technique (Rolfs et al., 1991). This technique requireschoosing pairs of oligonucleotide primers framing the fragment whichmust be amplified. Reference may, for example, be made to the techniquedescribed in U.S. Pat. No. 4,683,202. The amplified fragments can beidentified, for example after agarose or polyacrylamide gelelectrophoresis, or after a chromatographic technique such as gelfiltration or ion exchange chromatography, and then sequenced. Thespecificity of the amplification can be controlled using the nucleotidesequences of polynucleotides of the invention as primers, and plasmidscontaining these sequences, or else the derived amplification products,as matrices. The amplified nucleotide fragments can be used as reagentsin hybridization reactions in order to demonstrate the presence, in abiological sample, of a target nucleic acid of sequence complementary tothat of said amplified nucleotide fragments.

The invention is also directed toward the nucleic acids which can beobtained by amplification using primers according to the invention.

Other techniques amplifying the target nucleic acid can advantageouslybe employed as an alternative to PCR (PCR-like) using a pair of primersof nucleotide sequences according to the invention. The term “PCR-like”is intended to denote all the methods which use direct or indirectreproductions of nucleic acid sequences, or else in which the labelingsystems have been amplified; these techniques are of course known. Ingeneral, they involve amplification of the DNA with a polymerase; whenthe sample of origin is an RNA, a reverse transcription should becarried out beforehand. A very large number of methods currently existfor this amplification, such as, for example, the SDA (StrandDisplacement Amplification) technique (Walker et al., 1992), the TAS(Transcription-based Amplification System) technique described by Kwohet al. (1989), the 3SR (Self-Sustained Sequence Replication) techniquedescribed by Guatelli et al. (1990), the NASBA (Nucleic Acid SequenceBased Amplification) technique described by Kievitis et al. (1991), theTMA (Transcription Mediated Amplification) technique, the LCR (LigaseChain Reaction) technique described by Landegren et al. (1988), the RCR(Repair Chain Reaction) technique described by Segev (1992), the CPR(Cycling Probe Reaction) technique described by Duck et al. (1990), andthe Q-beta-replicase amplification technique described by Miele et al.(1983). Some of these techniques have since been improved.

When the target polynucleotide to be detected is an mRNA, an enzyme ofthe reverse transcriptase type is advantageously used, prior to carryingout an amplification reaction using the primers according to theinvention or to carrying out a method of detection using the probes ofthe invention, in order to obtain a cDNA from the mRNA contained in thebiological sample. The cDNA obtained will then serve as a target for theprimers or the probes used in the method of amplification or ofdetection according to the invention.

The probe hybridization technique can be carried out in various ways(Matthews et al., 1988). The most general method consists inimmobilizing the nucleic acid extracted from the cells of varioustissues or from cells in culture, on a support (such as nitrocellulose,nylon or polystyrene), and in incubating, under well-defined conditions,the immobilized target nucleic acid with the probe. After hybridization,the excess probe is removed and the hybrid molecules formed are detectedby the appropriate method (measuring the radioactivity, the fluorescenceor the enzyme activity associated with the probe).

According to another embodiment of the nucleic acid probes according tothe invention, the latter can be used as capture probes. In this case, aprobe termed “capture probe” is immobilized on a support and is used tocapture, by specific hybridization, the target nucleic acid obtainedfrom the biological sample to be tested, and the target nucleic acid isthen detected by virtue of a second probe, termed “detection probe”,labeled with a readily detectable element.

Among the advantageous nucleic acid fragments, it is thus necessary tomention, in particular, antisense oligonucleotides, i.e.oligonucleotides the structure of which ensures, by hybridization withthe target sequence, inhibition of the expression of the correspondingproduct. It is also necessary to mention sense oligonucleotides which,by interaction with proteins involved in regulating the expression ofthe corresponding product, will induce either inhibition or activationof this expression.

Such oligonucleotides can be used as therapeutic products and medicinalproducts for regulating the phenomena of apoptosis and of tumorsuppression. The use of these sense or antisense sequences can also beimplemented in vitro, to define novel means of modeling and of studyingapoptosis.

The present invention also relates to an isolated polypeptide,characterized in that it comprises a polypeptide chosen from:

-   -   a) a polypeptide of sequence SEQ ID No. 2;    -   b) a polypeptide which is a variant of a polypeptide of sequence        defined in a);    -   c) a polypeptide homologous to a polypeptide defined in a) or        b), comprising at least 80% identity with said polypeptide of        a);    -   d) a fragment of at least 15 consecutive amino acids of a        polypeptide defined in a), b) or c);    -   e) a biologically active fragment of a polypeptide defined in        a), b) or c).

For the purpose of the present invention, the term “polypeptide” isintended to denote proteins or peptides.

The term “biologically active fragment” is intended to mean a fragmenthaving the same biological activity as the peptide fragment from whichit is deduced, preferably within the same order of magnitude (to withina factor of 10). Thus, the examples show that the TSIP4 protein (SEQ IDNo. 2) has a potential role in the phenomena of apoptosis. Abiologically active fragment of the TSIP4 protein therefore consists ofa polypeptide derived from SEQ ID No. 2, also having a role inapoptosis.

Preferably, a polypeptide according to the invention is a polypeptideconsisting of the sequence SEQ ID No. 2 (corresponding to the proteinencoded by the TSIP4 gene) or of a sequence having at least 80% identitywith SEQ ID No. 2 after optimal alignment.

The sequence of the polypeptide exhibits a percentage identity of atleast 80%, after optimal alignment, with the sequences SEQ ID No. 2,preferably 90 or 95%, more preferably 98%, or 99%.

The expression “polypeptide the amino acid sequence of which exhibits apercentage identity of at least 80%, preferably 90%, more preferably98%, after optimal alignment, with a reference sequence” is intended todenote the polypeptides exhibiting certain modifications compared to thereference polypeptide, such as in particular one or more deletionsand/or truncations, an extension, a chimeric fusion, and/or one or moresubstitutions.

Among the polypeptides the amino acid sequence of which exhibits apercentage identity of at least 80%, preferably 90%, more preferably98%, after optimal alignment, with the sequence SEQ ID No. 2, or withone of its fragments, according to the invention, preference is given tothe variant polypeptides encoded by the variant nucleic acid sequencesas defined above, in particular the polypeptides the amino acid sequenceof which exhibits at least one mutation corresponding in particular to atruncation, deletion, substitution and/or addition of at least one aminoacid residue compared to the sequence SEQ ID No. 2 or with one of itsfragments, more preferably the variant polypeptides exhibiting amutation associated with a pathological condition, such as a cancer or aneurodegenerative disease.

The present invention also relates to the cloning and/or expressionvectors comprising a nucleic acid or encoding a polypeptide according tothe invention. Such a vector can also contain the elements required forthe expression and, optionally, for the secretion of the polypeptidein/from a host cell. Such a host cell is also a subject of theinvention.

The vectors characterized in that they comprise a promoter and/orregulator sequence according to the invention are also part of theinvention.

Said vectors preferably comprise a promoter, translation initiation andtermination signals, and also suitable regions for regulatingtranscription. It must be possible for them to be maintained stably inthe cell and they can optionally have particular signals specifying thesecretion of the translated protein.

These various control signals are chosen as a function of the cellularhost used. To this effect, the nucleic acid sequences according to theinvention can be inserted into vectors which replicate autonomously inthe chosen host, or vectors which integrate in the chosen host.

Among the autonomously replicating systems, use is preferably made,depending on the host cell, of systems of the plasmid or viral type, theviral vectors possibly being in particular adenoviruses (Perricaudet etal., 1992), retroviruses, lentiviruses, poxviruses or herpes viruses(Epstein et al., 1992). Those skilled in the art are aware of thetechnology which can be used for each of these systems.

When integration of the sequence into the host cell's chromosomes isdesired, use may, for example, be made of systems of the plasmid orviral type; such viruses are, for example, retroviruses (Temin, 1986) orAAVs (Carter, 1993).

Among the nonviral vectors, preference is given to naked polynucleotidessuch as naked DNA or naked RNA according to the technique developed bythe company VICAL, bacterial artificial chromosomes (BACs), yeastartificial chromosomes (YACs) for expression in yeast, mouse artificialchromosomes (MACs) for expression in murine cells and, preferably, humanartificial chromosomes (HACs) for expression in human cells.

Such vectors are prepared according to the methods commonly used bythose skilled in the art, and the clones resulting therefrom can beintroduced into a suitable host by standard methods, such as, forexample, lipofection, electroporation, heat shock, transformation afterchemical permeabilization of the membrane, or cell fusion.

The invention also comprises the host cells, in particular theeukaryotic or prokaryotic cells, transformed with the vectors accordingto the invention, and also the transgenic animals, preferably mammals,except humans, comprising one of said transformed cells according to theinvention. These animals can be used as models, for studying theetiology of inflammatory and/or immune diseases, and in particularinflammatory diseases of the digestive tract, or for studying cancers.

Among the cells which can be used for the purposes of the presentinvention, mention may be made of bacterial cells (Olins and Lee, 1993),but also yeast cells (Buckholz, 1993), as well as animal cells, inparticular mammalian cell cultures (Edwards and Aruffo, 1993), andespecially chinese hamster ovary (CHO) cells. Mention may also be madeof insect cells in which use may be made of methods using, for example,baculoviruses (Luckow, 1993). A preferred cellular host for expressionof the proteins of the invention consists of COS cells.

Among the mammals according to the invention, preference is given toanimals such as rodents, in particular mice, rats or rabbits, expressinga polypeptide according to the invention.

Among the mammals according to the invention, preference is also givento animals such as mice, rats or rabbits, characterized in that the geneencoding the protein of sequence SEQ ID No. 2, or the sequence of whichis encoded by the homologous gene in these animals, is not functional,is knocked out, or exhibits at least one mutation.

These transgenic animals are obtained, for example, by homologousrecombination on embryonic stem cells, transfer of these stem cells toembryos, selection of the chimeras affected in the reproductive lines,and growth of said chimeras.

The transgenic animals according to the invention can thus overexpressthe gene encoding the protein according to the invention, or theirhomologous gene, or express said gene into which a mutation isintroduced. These transgenic animals, in particular mice, are obtained,for example, by transfection of the copy of this gene under the controlof a strong promoter which is ubiquitous in nature, or selective for atissue type, or after viral transcription.

Alternatively, the transgenic animals according to the invention can bemade deficient for the gene encoding the polypeptide of sequence SEQ IDNo. 2, or its homologous gene, by inactivation using the LOXP/CRErecombinase system (Rohlmann et al., 1996) or any other system ofinactivation of the expression of this gene.

The cells and mammals according to the invention can be used in a methodfor producing a polypeptide according to the invention, as describedbelow, and can also be used as an analytical model.

The transformed cells or mammals as described above can also be used asmodels in order to study the interactions between the polypeptidesaccording to the invention, and the chemical or protein compoundsinvolved directly or indirectly in the activities of the polypeptidesaccording to the invention, in order to study the various mechanisms andinteractions which come into play.

They can in particular be used to select products which interact withthe polypeptides according to the invention, in particular the proteinof sequence SEQ ID No. 2 or its variants according to the invention, asa cofactor or as an inhibitor, in particular a competitive inhibitor, orelse which have agonist or antagonist activity with respect to theactivity of the polypeptides according to the invention. Preferably,said transformed cells or transgenic animals are used as a model inparticular for selecting products for combating pathological conditionsassociated with abnormal expression of this gene.

The invention also relates to the use of a cell, of a mammal or of apolypeptide according to the invention, for screening chemical orbiochemical compounds which can interact directly or indirectly with thepolypeptides according to the invention, and/or which are capable ofmodulating the expression or the activity of these polypeptides.

Similarly, the invention also relates to a method for screeningcompounds capable of interacting, in vitro or in vivo, with a nucleicacid according to the invention, using a nucleic acid, a cell or amammal according to the invention, and detecting the formation of acomplex between the candidate compounds and the nucleic acid accordingto the invention.

The compounds thus selected are also subjects of the invention.

Such a compound according to the invention may be a compound which has achemical structure, a lipid, a sugar, a protein, a peptide, aprotein-lipid, protein-sugar, peptide-lipid or peptide-sugar hybridcompound, or a protein or a peptide to which chemical branches have beenadded.

Among the chemical compounds envisaged, they may contain one or morerings, which may or may not be aromatic, and also several residues ofany type (in particular lower alkyl, i.e. exhibiting between 1 and 6carbon atoms).

The invention also relates to the use of a nucleic acid sequenceaccording to the invention, for synthesizing recombinant polypeptides.

The method for producing a polypeptide according to the invention inrecombinant form, which is itself included in the present invention, ischaracterized in that the transformed cells, in particular the mammaliancells of the present invention, are cultured under conditions whichallow the expression of a recombinant polypeptide encoded by a nucleicacid sequence according to the invention, and in that said recombinantpolypeptide is recovered.

The recombinant polypeptides, characterized in that they can be obtainedusing said method of production, are also part of the invention.

The recombinant polypeptides obtained as indicated above can be in bothglycosylated and nonglycosylated form, and may or may not exhibit thenatural tertiary structure.

The sequences of the recombinant polypeptides can also be modified inorder to improve their solubility, in particular in aqueous solvents.

Such modifications are known to those skilled in the art, such as, forexample, the deletion of hydrophobic domains or the substitution ofhydrophobic amino acids with hydrophilic amino acids.

These polypeptides can be produced from the nucleic acid sequencesdefined above, according to the techniques of recombinant polypeptideproduction known to those skilled in the art. In this case, the nucleicacid sequence used is placed under the control of signals which allowits expression in a cellular host.

An effective system for producing a recombinant polypeptide requireshaving a vector and a host cell according to the invention.

These cells can be obtained by introducing into host cells a nucleotidesequence inserted into a vector as defined above, and then culturingsaid cells under conditions which allow the replication and/or theexpression of the transfected nucleotide sequence.

The methods used to purify a recombinant polypeptide are known to thoseskilled in the art. The recombinant polypeptide can be purified fromcell lysates and extracts, from the culture medium supernatant, bymethods used individually or in combination, such as fractionation,chromatography methods, immunoaffinity techniques using specificmonoclonal or polyclonal antibodies, etc.

The polypeptides according to the present invention can also be obtainedby chemical synthesis using one of the many known peptide syntheses, forexample the techniques using solid phases (see in particular Stewart etal., 1984) or techniques using partial solid phases, by fragmentcondensation or by conventional synthesis in solution.

The polypeptides which are obtained by chemical synthesis and which cancomprise corresponding unatural amino acids are also included in theinvention.

The mono- or polyclonal antibodies or their fragments, chimericantibodies or immunoconjugated antibodies, characterized in that theyare capable of specifically recognizing a polypeptide according to theinvention, are part of the invention.

Specific polyclonal antibodies can be obtained from a serum of an animalimmunized against the polypeptides according to the invention, inparticular produced by genetic recombination or by peptide synthesis,according to the usual procedures.

The advantage of antibodies which specifically recognize certainpolypeptides, or variants, or their immunogenic fragments, according tothe invention is in particular noted.

The mono- or polyclonal antibodies or their fragments, chimericantibodies or immunoconjugated antibodies, characterized in that theyare capable of specifically recognizing the polypeptide of sequence SEQID No. 2, are particularly preferred.

The specific monoclonal antibodies can be obtained according to theconventional method of hybridoma culture described by Kohler andMilstein (1975).

The antibodies according to the invention are, for example, chimericantibodies, humanized antibodies, or Fab or F(ab′)₂ fragments. They canalso be in the form of immunoconjugates or of antibodies which arelabeled in order to obtain a detectable and/or quantifiable signal.

The invention also relates to methods for detecting and/or purifying apolypeptide according to the invention, characterized in that they usean antibody according to the invention.

The invention also comprises purified polypeptides, characterized inthat they are obtained using a method according to the invention.

Moreover, besides their use for purifying the polypeptides, theantibodies of the invention, in particular the monoclonal antibodies,can also be used for detecting these polypeptides in a biologicalsample.

They thus constitute a means of immunocytochemical orimmunohistochemical analysis of the expression of the polypeptidesaccording to the invention, in particular the polypeptide of sequenceSEQ ID No. 2 or one of its variants, on specific tissue sections, forexample by immunofluorescence or gold labeling, or with enzymeimmunoconjugates.

They can in particular make it possible to demonstrate abnormalexpression of these polypeptides in the tissues or biological specimens.

More generally, the antibodies of the invention can advantageously beused in any situation where the expression of a normal or mutatedpolypeptide according to the invention must be observed.

Thus, a method for detecting a polypeptide according to the invention ina biological sample, comprising the steps of bringing the biologicalsample into contact with an antibody according to the invention anddemonstrating the antigen-antibody complex formed, is also a subject ofthe invention, as is a kit for implementing such a method. Such a kitcontains in particular:

-   -   a) a monoclonal or polyclonal antibody according to the        invention;    -   b) optionally, reagents for constituting a medium suitable for        the immunoreaction;    -   c) the reagents for detecting the antigen-antibody complex        produced during the immunoreaction.

The antibodies according to the invention can also be used in thetreatment of a neurodegenerative disease or of a cancer, in humans, whenabnormal expression of the TSIP4, PS1 or p53. gene, or a gene describedby the inventors of the present application in the application WO97/22695 or WO 00/08147, is observed. An abnormal expression means anoverexpression, an underexpression or the expression of a mutatedprotein.

These antibodies can be obtained directly from human serum, or can beobtained from animals immunized with polypeptides according to theinvention, and then “humanized”, and can be used as they are or in thepreparation of a medicinal product intended for the treatment of theabovementioned diseases.

The methods for determining an allelic variability, a mutation, adeletion, a loss of heterozygosity, or any genetic abnormality of thegene according to the invention, characterized in that they use anucleic acid sequence, a polypeptide or an antibody according to theinvention, are also part of the invention.

It is possible to detect the mutations in the sequence of the TSIP4 genesuppressed during p53-induced apoptosis, directly by analysis of thenucleic acid and of the sequences according to the invention (genomicDNA, RNA or cDNA), but also by the polypeptides according to theinvention. In particular, the use of an antibody according to theinvention which recognizes an epitope carrying a mutation makes itpossible to discriminate between a “healthy” protein and a protein“associated with a pathological condition”.

The precise description of the mutations which can be observed in theTSIP4 gene can thus make it possible to form the bases of a moleculardiagnosis for neurodegenerative diseases and cancers, or any diseaseinvolving apoptosis. Such an approach, based on searching for mutationsin the gene, will make it possible to contribute to the diagnosis ofthese diseases and, optionally, will make it possible to reduce theextent of certain additional, invasive or expensive, examinations. Theinvention forms the bases of such a molecular diagnosis based on thesearch for mutations in TSIP4.

In particular, preference is given to a method of diagnosis and/or ofprognostic evaluation of a neurodegenerative disease or of a cancer,characterized in that the presence of at least one mutation and/or analteration of expression of the gene corresponding to SEQ ID No. 1 isdetermined, using a biological specimen from a patient, by analyzing allor part of a nucleic acid sequence corresponding to said gene.

This method of diagnosis and/or of prognostic evaluation can be usedpreventatively (study of a predisposition to a neurodegenerative diseaseor to cancer), or to serve in establishing and/or confirming a clinicalcondition in a patient.

Preferably, the neurodegenerative disease is Alzheimer's disease.

The analysis can be carried out by sequencing all or part of the gene,or by other methods known to those skilled in the art. Methods based onPCR, for example PCR-SSCP, which makes it possible to detect pointmutations, can in particular be used.

The analysis can also be carried out by attachment of a probe accordingto the invention, corresponding to one of the sequences SEQ ID No. 1, toa DNA chip and hybridization on these microplates. A DNA chip containinga sequence according to the invention is also one of the subjects of theinvention.

Similarly, a protein chip containing an amino acid sequence according tothe invention is also a subject of the invention. Such a protein chipmakes it possible to study the interactions between the polypeptidesaccording to the invention and other proteins or chemical compounds, andcan thus be of use for screening compounds which interact with thepolypeptides according to the invention. The protein chips according tothe invention can also be used to detect the presence of antibodiesdirected against the polypeptides according to the invention, in theserum of patients. A protein chip containing an antibody according tothe invention can also be used.

Those skilled in the art also know how to use techniques for studyingthe alteration of the expression of a gene, for example by studying themRNA (in particular by Northern Blotting or by RT-PCR experiments, withprobes or primers according to the invention), or the protein expressed,in particular by Western Blotting using antibodies according to theinvention.

The invention also relates to methods for obtaining an allele of theTSIP4 gene, associated with a detectable phenotype, comprising thefollowing steps:

-   -   a) obtaining a nucleic acid sample from an individual expressing        said detectable phenotype;    -   b) bringing said nucleic acid sample into contact with an agent        capable of specifically detecting a nucleic acid encoding the        TSIP4 protein;    -   c) isolating said nucleic acid encoding the TSIP4 protein.

Such a method can be followed by a step of sequencing all or part of thenucleic acid encoding the TSIP4 protein, which makes it possible topredict the susceptibility to a neurodegenerative disease or to acancer.

The agent capable of specifically detecting a nucleic acid encoding theTSIP4 protein is advantageously an oligonucleotide probe according tothe invention, which can be made up of DNA, RNA or PNA, which may or maynot be modified. The modifications may include radioactive orfluorescent labeling, or may be due to modifications in the bondsbetween the bases (phosphorothioates or methylphosphonates for example).Those skilled in the art are aware of the protocols for isolating aspecific DNA sequence. Step b) of the method described above may also bean amplification step as described previously.

The invention also relates to a method for detecting and/or assaying anucleic acid according to the invention in a biological sample,comprising the following steps of bringing a probe according to theinvention into contact with a biological sample and detecting and/orassaying the hybrid formed between said polynucleotide and the nucleicacid of the biological sample.

Those skilled in the art know how to implement such a method, and can inparticular use a reagent kit comprising:

-   -   a) a polynucleotide according to the invention, used as a probe;    -   b) the reagents required to carry out a hybridization reaction        between said probe and the nucleic acid of the biological        sample;    -   c) the reagents required for detecting and/or assaying the        hybrid formed between said probe and the nucleic acid of the        biological sample;        which is also a subject of the invention.

Such a kit can also contain positive or negative controls in order toensure the quality of the results obtained.

However, in order to detect and/or assay a nucleic acid according to theinvention, those skilled in the art may also carry out an amplificationstep using primers chosen from the sequences according to the invention.

Finally, the invention also relates to the compounds chosen from anucleic acid, a polynucleotide, a vector, a cell or an antibodyaccording to the invention, or the compounds obtained using thescreening methods according to the invention, as a medicinal product, inparticular for preventing and/or treating a neurodegenerative disease ora cancer, associated with the presence of at least one mutation of thegene corresponding to SEQ ID No. 1, and/or with abnormal expression ofthe protein corresponding to SEQ ID No. 2.

The present invention also relates to methods for screening andidentifying products which can interfere in the p53 cascade, and thusinduce tumor reversion and/or apoptosis or, conversely, decrease thephenomena of apoptosis.

This aspect of the present invention is based on the fact that the TSIP4protein, which is the subject of the present invention, binds to theTSIP2 protein described in patent application WO 97/22695, the role ofwhich in Alzheimer's disease has been reported.

In a first embodiment, the invention is directed toward a method forscreening, selecting or identifying a compound which interferes with,decreases or inhibits the binding of TSIP4 to TSIP2, having the stepsof:

-   -   a) bringing said compound into contact with a system for        determining, in vitro, the binding between TSIP4 and TSIP2;    -   b) identifying the decrease and/or the inhibition of the binding        between TSIP4 and TSIP2.

A subject of the present invention is also in particular a method forscreening, selecting or identifying a compound for decreasing and/orinhibiting tumor suppression and/or cell death (apoptosis), having thesteps of:

-   -   a) bringing compounds into contact with a system for        determining, in vitro, the binding between TSIP4 and TSIP2;    -   b) identifying the compounds which induce the decrease and/or        the inhibition of the binding between TSIP4 and TSIP2;    -   c) studying the compounds identified in step b) in a model for        measuring the phenomena of apoptosis and/or tumor suppression;    -   d) identifying the decrease and/or the inhibition in tumor        suppression and/or cell death in said model compared to a        control model with which said compound has not been brought into        contact.

Some models for studying/measuring apoptosis and/or tumor suppressionhave already been described, and consist in particular of the in vitromodels described by Tellerman et al. (1993, Proc. Natl. Acad. Sci. USA,90, 8702-6), Amson et al. (1996, Proc. Natl. Acad. SCI; USA, 93, 3953-7)or Nemani et al. (1996, Proc. Natl. Acad. Sci. USA, 93, 9039-42). Inorder to determine compounds which make it possible to increase tumorsuppression and/or cell death (apoptosis), it is also possible tocombine various methods according to the invention; it is in particularpossible to thus obtain a method for screening, selecting or identifyingcompounds whose function is an increase in tumor suppression and/or celldeath (apoptosis), having the steps of:

-   -   a) bringing said compound into contact with a system for        determining, in vitro, the binding between TSIP4 and TSIP2;    -   b) identifying the compounds which induce the decrease and/or        the inhibition in the binding between TSIP4 and TSIP2;    -   c) bringing the compounds selected in step b) into contact in a        model for measuring the phenomena of apoptosis and/or tumor        suppression;    -   d) identifying the increase in tumor suppression and/or cell        death in said model compared to a control model with which said        compound has not been brought into contact.

Such a method is therefore also a subject of the present invention.

The present invention therefore uses the fact that the TSIP4 and TSIP2proteins can bind to one another. It is therefore advantageous toidentify the domains of each protein which are effectively in contactwith the other protein. As a result, this should make it possible to beable to use the peptides thus identified as decoys or agonists for thecomplete proteins. This may thus make it possible to define compoundswhich will interfere in the TSIP4-TSIP2 binding, and which may possiblyinduce tumor suppression and/or apoptosis or, conversely, decrease thesephenomena.

More generally, the present invention makes it possible to identifyregions of TSIP4 involved in binding with the TSIP2 protein, using amethod comprising the steps of:

-   -   a) bringing peptides derived from the TSIP4 protein into contact        in a system for determining, in vitro, the binding between TSIP4        and TSIP2;    -   b) identifying the peptides which bring about the decrease in        binding between TSIP4 and TSIP2 in said system.

It is obvious that the present invention also makes it possible todetermine the regions of TSIP2 involved in the binding with TSIP4,according to methods similar to the methods described above, and thatthese regions can in particular be used as decoys when the intention isto decrease tumor suppression and/or apoptosis.

The term “region” of a protein is in particular intended to meanpeptides of primary sequence derived from the primary sequence of saidprotein.

These regions thus identified can be tested, like the products describedabove, for their properties in the regulation of tumor suppressionand/or apoptosis.

The present invention therefore makes it possible to identify productswhich make it possible to interact with the TSIP4-TSIP2 binding, andwhich therefore may be of value in the regulation of apoptosis and/ortumor suppression. However, it is possible that these products, in orderto be able to be used for a therapeutic treatment, in particular canceror neurodegenerative diseases, require optimization in order to havegreater activity and/or less toxicity.

Specifically, the development of a medicinal product is often carriedout according to the following principle:

-   -   screening compounds having a desired activity using a suitable        method,    -   selecting the compounds which satisfy the “specifications”,    -   determining the structure (in particular the sequence        (optionally tertiary sequence) if they are peptides, formula and        backbone if they are chemical compounds) of the compounds        selected,    -   optimizing the compounds selected, by modification of the        structure (for example by changing the stereochemical        conformation (for example switching from L to D for the amino        acids in a peptide), adding substituents to the peptide or        chemical backbones, in particular by grafting residues onto the        backbone, modification of the peptides (see in particular Gante        (“Peptidomimetics”, in Angewandte Chemie-International Edition        Engl. 1994, 33. 1699-1720))),    -   passing and screening the compounds thus obtained on suitable        models which are often models closer to the pathology studied.        At this stage, animal models are in particular often used, in        general in rodents (mice, rats, etc.) or in dogs, or even        primates.

The animal models which can be used are, for example, for cancer, modelsbased on immunodepressed mice (for example scid/scid), into which tumorcells, which will lead to the development of tumors, are injected (inparticular subcutaneously). The effectiveness of the potentiallyantitumor compounds is studied, for example by measuring the size of thetumors formed.

To study neurodegenerative diseases, the model described by Amson et al.(2000, Proc. Natl. Acad. Sci. USA, 97, 5346-50), which consists ofp53-deficient mice, or the model described in Chen et al., Janus et al.,and Morgan et al. (2000, Nature, 408 pp. 975-985), can be used.

Thus, an object of the present invention is in particular to make itpossible to identify compounds which might be used for the treatment ofcancer, in that they have an activity of increasing tumor suppressionand/or apoptosis. One of the subjects of the present invention istherefore a method comprising the steps of:

-   -   a) implementing a method according to the invention, for        identifying compounds having a certain activity of increasing        tumor suppression and/or apoptosis,    -   b) modifying the product selected in step a),    -   c) testing the product modified in step b) in in vitro and/or in        vivo methods, on relevant models of tumor suppression and/or        apoptosis,    -   d) identifying the product which makes it possible to obtain an        activity of tumor suppression and/or apoptosis greater than the        activity obtained for the product selected in step a).

Step d) can be replaced with a step d′), which would be:

-   -   d′) identifying the product which makes it possible to obtain        the desired biological effect with less toxicity in an animal        model (when one of the models used in step c) is in vivo).

When the tests are carried out on models of apoptosis or of tumorsuppression in vitro, it is possible, for example, to use the modelK256/KS described by Tellerman et al. (1993, Proc. Natl. Acad. Sci. USA,90, 8702-6). It is also possible to use the M1-LTR cells described byAmson et al. (1996, Proc. Natl. Acad. SCI; USA, 93, 3953-7), or theU937/US3-US4 cells described by Nemani et al. (1996, Proc. Natl. Acad.Sci. USA, 93, 9039-42).

The in vivo trials can be carried out by injecting these cells intoanimals, in particular immunodepressed mice and studying the effects ofthe various compounds tested.

Those skilled in the art will be able to define the conditions and thethresholds necessary for identifying a product which can be used as amedicinal product, according to the regulatory requirements (inparticular for toxicology), with respect to the benefit provided by theproduct thus identified.

In the same way, the invention also relates to the methods foroptimizing the products which suppress tumor suppression and/orapoptosis, identified using the methods described above, and which makeit possible to identify products which can be used as medicinalproducts.

Thus, the invention also relates to a method for identifying a producthaving an activity of decreasing and/or inhibiting tumor suppressionand/or apoptosis, characterized in that it comprises the steps of:

-   -   a) implementing a method according to the invention, for        identifying compounds having a certain activity of decreasing        tumor suppression and/or apoptosis,    -   b) modifying the product selected in step a), in particular by        grafting residues onto the chemical backbone,    -   c) testing the product modified in step b) in in vitro and/or in        vivo methods, on relevant models of tumor suppression and/or        apoptosis,    -   d) identifying the product which makes it possible to obtain an        activity of tumor suppression and/or apoptosis which is        decreased compared to the activity obtained for the product        selected in step a).

Step d) can also be replaced with a step d′), which would be:

-   -   d′) identifying the product which makes it possible to obtain        the desired biological effect with less toxicity in an animal        model (when one of the models used in step c) is in vivo).

In fact, this also involves being able to obtain the product whichexhibits the best (biological activity and clinical effect)/(potentialrisks for use) ratio.

The parameters to be brought into play in order to obtain these resultsare all known and within the scope of those skilled in the art who wishto develop novel medicinal products, and can be found, for example, inthe directives of the organizations such as the l'Agence du Médicament[French Drug Agency], European Commission or Federal Drug Agency.

The use of the methods according to the present invention requiresmodels which make it possible to determine the binding between TSIP4 andTSIP2, and also an easy measurement of the increases or decrease in theamount of TSIP2 or p53 protein in a eukaryotic cell.

The amount of TSIP2 or p53 protein can be studied by Western Blotting,the revelation being carried out using an antibody directed against saidprotein. Such antibodies, directed against p53, can in particular befound at Calbiochem, under the reference OP09L.

In order to implement the methods according to the invention requiringstudying and screening on mammalian cells, it may be advantageous tooverexpress one or other of the TSIP4, TSIP2 and p53 proteins in saidcells, two of them, or all three together.

Thus, it is easier to be able to study the variations in the amount ofTSIP2 protein or of p53, in particular by comparison of the cells onwhich the compounds of interest are tested with the same cells, to whichthe compounds which it is desired to screen are not added.

It is understood that the expression of the two proteins TSIP4 andTSIP2, and p53, can be increased by introducing the genes (in particularthe cDNAs) encoding these proteins into the cells, either placed onvectors or by introduction into the chromosome.

When episomal expression is chosen, said mammalian cell is transfectedwith at least one vector chosen from a vector carrying a DNA fragmentencoding TSIP4, a vector carrying a DNA fragment encoding TSIP2, avector carrying a DNA fragment encoding p53, and a vector carrying a DNAfragment encoding more than one of these proteins. Thus, the same vectorcan express all the proteins; alternatively, it is possible to introduceseveral vectors.

It is possible to use vectors which allow expression and easypurification of the TSIP4 and TSIP2 proteins, for example in prokaryoticcells (E. coli, B. subtilis etc.) or eukaryotic cells (yeast such asSaccharomyces, Kluyveromyces, etc.), mammalian cells (HeLa, Cos, Hep-2,etc.) or insect cells (using a Baculovirus system). Thus, it may beadvantageous for the proteins to have a tag at their N- or C-terminalend, in order to facilitate the purification. A histidine or GST tag isin particular chosen. These methods are well known to those skilled inthe art, who find the suitable plasmids in the catalogues of companiessuch as Stratagéne.

When the methods according to the invention are implemented on in vitromodels, to study the binding between TSIP2 and TSIP4, there are severalways to carry out the procedures.

A protocol which can be used may be as follows:

-   -   expression and purification of the TSIP4 and TSIP2 proteins, for        example in prokaryotic cells (E. coli, B. subtilis, etc.) or        eukaryotic cells (yeast such as Saccharomyces, Kluyveromyces,        etc.), mammalian cells (HeLA, Cos, Hep-2, etc.) or insect cells        (using a Baculovirus system). It may be advantageous for the        proteins to have a tag at their N- or C-terminal end, in order        to facilitate the purification. A histidine or GST tag is in        particular chosen. These methods are well known to those skilled        in the art, who find the suitable plasmids in the catalogues of        companies such as Stratagéne;    -   binding of the proteins to suitable beads. When a GST tag is        used, the expressed proteins are bound to sepharose beads        exhibiting glutathione;    -   preparation of proteins by in vitro translation. This can be        easily carried out using commercially available vectors (for        example available from Promega), which make it possible to clone        the cDNAs under the control of well-known promoters (T7 or T3)        and to use suitable RNA polymerases to produce the RNAs, and        then to effect the expression of the proteins in vitro, using        the available kits and following the manufacturer's indications;    -   coprecipitation of the proteins, by adding the proteins obtained        by in vitro translation to the sepharose-glutathione beads to        which the proteins from fusion with GST are attached. After a        sufficient amount of contact time, the beads are washed and an        analysis is carried out by SDS-PAGE gel electrophoresis and        autoradiography. The appearance of the bands corresponding to        the two proteins clearly shows binding between them.

The use of suitable controls thus makes it possible to define thedecrease and/or the inhibition of the binding between TSIP4 and TSIP2,by comparison of the amounts of proteins released after adding thecompound tested during the coprecipitation step, with the amounts ofproteins released in the controls.

It is also possible to study the binding of the proteins using the FRET(Fluorescence Resonance Energy Transfer) system, which consists inlabeling each one of the proteins with a suitable residue, the bindingof the two proteins inducing a reaction between each one of the tworesidues and the emission of a readily detectable fluorescence.

A subject of the present invention is also the compounds which can beobtained using a method according to the invention, in particular thecompounds which have an activity of increasing tumor suppression and/orapoptosis, those which have an activity of inhibiting the TSIP4-TSIP2binding, and also those which have an activity of decreasing and/orinhibiting tumor suppression and/or apoptosis.

The present invention also relates to the peptide sequencescorresponding to a region of TSIP4 which interacts with the TSIP2protein, which can in particular be identified using a method accordingto the invention.

The invention also relates to the peptide sequences corresponding to aregion of TSIP2 which interacts with the TSIP4 protein, which can inparticular be identified using a method according to the invention, themethod which makes it possible to identify the peptide sequences ofTSIP4 which interact with TSIP2 possibly being adapted to determine thepeptide sequences of TSIP2 which interact with TSIP4, in particular byadapting the in vitro protocol developed above.

The invention also relates to the nucleotide sequences encoding thepeptide sequences thus identified.

It is clear that the term “peptide sequence” or “nucleic acid sequence”or “nucleotide sequence” (the latter two terms being used indifferently)represents sequences which are isolated, i.e. outside of their naturalstate, and which can in particular be modified by replacing their baseunits with unnatural units, or by modifying the bonds between the baseunits (for example phosphorothioates (nucleic acid) or peptide nucleicacids).

An object of the invention is to make it possible to identify compoundswhich interfere with the TSIP4 and TSIP2 binding, some of thesecompounds possibly in particular inducing effects on the p53 cascade.Thus, the compounds according to the invention, the peptide sequencesaccording to the invention or the nucleotide sequences according to theinvention, as a medicinal product, are also subjects of the invention.

A compound identified using a method according to the invention may be acompound which has a chemical structure, a lipid, a sugar, a protein, apeptide, a protein-lipid, protein-sugar, peptide-lipid or peptide-sugarhybrid compound, or a protein or a peptide to which chemical brancheshave been added.

Among the chemical compounds envisioned, they may contain one or more(in particular two or three) rings, which may or may not be aromatic,having from three to eight carbon atoms, and also several residues ofany type (in particular lower alkyl, i.e. exhibiting between one to sixcarbon atoms).

These compounds, nucleic acid sequences and peptide sequences can thusbe used to prepare a medicinal product intended in particular for thetreatment of cancer or of a neurodegenerative disease, depending on thepro- or anti-apoptosis/tumor reversion effect.

The inventors of the present application have, for the first time,demonstrated the fact that the TSIP4 protein binds to the TSIP2 protein.Thus, the present invention also relates to a complex consisting of aTSIP4 protein and a TSIP2 protein.

The present invention also relates to a method for inhibiting thebinding of TSIP4 to TSIP2 in a cell, comprising the step of:

-   -   a) bringing said cell into contact with a compound identified        using a method according to the invention, which inhibits the        TSIP4-TSIP2 binding.

The compound thus envisioned can also be a “decoy” peptide derived fromthe TSIP4 protein or from the TSIP2 protein. The method can beimplemented in vitro or in vivo.

The present invention is also directed toward a method for the treatmentof a cancer, characterized in that a compound which has been identifiedaccording to the present invention and which increases apoptosis and/ortumor reversion is administered to a patient.

A method for the treatment of a neurodegenerative disease, consisting inadministering, to a patient, a compound according to the presentinvention which decreases or inhibits apoptosis, is also a subject ofthe present invention.

The following examples make it possible to understand more clearly theadvantages of the invention and should not be considered as limiting thescope of the invention.

DESCRIPTION OF THE FIGURES

FIG. 1: description of the TSIP4 mRNA in various tissues.

FIG. 2: differential expression of TSIP 4 in the K562/KS system.

EXAMPLES Example 1 Binding of TSIP4 and TSIP2

The binding existing between TSIP 4 and TSIP 2 was observed in adouble-hybrid system derived from the system developed by Finley andBrent (Interaction trap cloning with yeast, 169-203, in DNA Cloning,Expression Systems: a practical Approach, 1995, Oxford Universal Press,Oxford), using presenilin 1 (TSIP2) as bait and TSIP4 as prey.

The TSIP2 gene (cDNA) was cloned into the plasmid pEG202 known to thoseskilled in the art for such an application (promoter 67-1511, lexA1538-2227, ADH Ter 2209-2522, pBR remnants 2540-2889, 2μ ori 2890-4785,YSCNFLP 4923-5729, HIS3 7190-5699, TYIB 7243-7707, RAF_part 7635-7976,backbone pBR 7995-10166, bla 8131-8988).

The TSIP4 gene (cDNA) was cloned into the plasmid pJG4-5, also wellknown to those skilled in the art (promoter GAL 1-528, fusion cassette528-849, ADH Ter 867-1315, 2μ ori 1371-3365, TRP1 3365-4250, backbonepUC 4264-6422, Ap 4412-5274).

The reporter plasmid pSH18-34, also known to those skilled in the art,is also used. This plasmid is in particular available from Invitrogen,under the reference number V611-20, and is also already transformed intothe strain EGY48 (also called RFY 231), from the same supplier(reference strain alone: C835-00, transformed with pSH18-34: C836-00).

The binding was demonstrated in the yeast strain RFY 231 (described inFinley Jr, et al, 1998, Proc Natl Acad Sci USA, 95 14266-71). This yeaststrain has the genotype (MATα trplΔ::hisG his3 ura3-1leu2::3Lexop-LEU2), and is derived from EGY48 (Guris et al., 1993, Cell,75, 791-803).

The reporter gene was the LacZ gene.

The study is carried out on a medium containing galactose and notcontaining leucine, and the presence of colored colonies on these dishesis studied.

It was thus possible to show that the TSIP21 protein binds to the TSIP4protein, and that the binding is on the 204 C-terminal amino acids ofthe TSIP2 protein.

Example 2 Analysis of the TSIP4 Protein

It appears that the TSIP 4 protein has two alternative forms, onecorresponding to SEQ ID No. 2, encoded by SEQ ID No. 1, the othercorresponding to amino acids 198-400 of SEQ ID No. 2, encoded by bases592 to 1200 of SEQ ID No. 1. A putative signal peptide corresponds toamino acids 198-243 of SEQ ID No. 2 (bases 592 to 729 of SEQ ID No. 1).

The sequences described above are also subjects of the invention.

The (complete) TSIP4 protein has 4 predicted transmembrane domains(amino acids 224-244, 260-280, 320-340, and 350-370), the N- andC-terminal ends being located in the cytoplasm. These transmembranedomains, and also the intra- or extracellular domains, are also subjectsof the invention, as are the sequences which encode them.

Example 3 Expression of the TSIP 4 Protein

Hybridization of a Northern Blot of various human tissues with a probecorresponding to a partial sequence of TSIP4 shows the existence of twotranscripts (approximately 1.8 and 6 kb). The mRNA is found mainly inthe heart, the skeletal muscle and the brain. It is also present, to alesser extent, in the placenta, the pancreas and the kidney. Itsexpression is very low in the lung and virtually nonexistent in theliver (FIG. 1).

Example 4 Differential Expression of TSIP4 in the K562/KS System

Hybridization of a Northern Blot with the TSIP4 probe showed a decreasedsignal compared to the K562 line, whereas an equivalent signal isobtained with the control GAPDH probe (FIG. 2). The K562/KS model wasdescribed by Telerman et al. (1993, Proc. Natl. Acad. Sci. USA, 90,8702-6).

The TSIP4 protein is therefore suppressed in a model of tumorsuppression.

Example 5 Expression of GST Fusion proteins

To obtain GST fusion proteins, the protocol below can be followed:

Preparation of a preculture from an isolated colony of B121 (DE3),transformed with plasmid pGEX-P-1-TSIP4 or pGEX-P-1-TSIP2, in an SBmedium with 100 μg/ml of ampicillin, at 37° C.

The plasmids are available from Amersham Pharmacia Biotech AB.

The proteins are the human proteins, encoded by the complementary cDNAscorresponding to the sequences SEQ ID No. 1 (TSIP4) and the completesequence of TSIP2 (GenBank U50957).

The following day, 250 ml of SB+Amp are inoculated with 5 ml of thepreculture.

Growth is at 28° C. or 37° C. depending on the toxicity of the proteinsfor the host bacteria, until an optical density of between 0.5 and 0.7is reached.

0.1 mM IPTG is added to induce the protein synthesis.

Growth is at 28° C. or 37° C. for 1 h or 1 h 30.

Centrifugation is carried out at 3000 rpm for 10 min (1800 g, 4° C.).

The precipitate is resuspended in 10 ml of buffer A NP40 (1% of NP40; 10mM Tris, pH 7.4; 150 mM NaCl; 1 mM EDTA; 10% glycerol; 1 mM DTT; 2 pg/mlaprotinin; 2 μg/ml leupeptin; 2 μg/ml pepstatin; 1 mM AEBSF).

Sonication is carried out 3 times for 15 s at power 50, on ice.

Centrifugation is carried out at 12000 rpm for 10 min (18000 g, 4° C.).

The supernatant is kept at −80° C.

Example 6 Protocol to be Followed for Binding of the Fusion Proteins tothe Sepharose-Glutathione Beads

2 ml of supernatant are added to 200 μl of beads (prepared after 3rinses in PBS, 1 rinse in the buffer A NP40, resuspension at 50% (weightby volume) in the buffer A NP40, centrifugation at 3000 rpm each time).

The glutathione-sepharose 4B beads are available from Amersham PharmaciaBiotech AB, under the number 17.0756.01.

Gentle mixing is carried out for at least 1 hour at 4° C.

The beads are rinsed 3 times in the buffer A NP40 without proteaseinhibitor.

The beads are resuspended in 1 ml of buffer A NP40 with proteaseinhibitor.

For the analysis by SDS-PAGE electrophoresis, a 20 to 40 μl sample ofthe resuspended beads is taken and centrifuged for 5 min, thesupernatant is discarded, the beads are resuspended in 10 μl of loadingbuffer X, and heating is carried out at 97° C. for 7 min. The gel isloaded and analyzed after revelation with Coomassie blue, in order tostandardize the amount of the fusion proteins to be used.

Example 7 Protocol for the in vitro Protein Translation

The TNT Coupled Reticulocyte Lysate System kit from Promega is used withthe T7 or T3 RNA polymerases, depending on the vector used to translateand express the proteins (T7 for AIPI and TSIP4 1, T3 for TSIP2) The kitis used according to the manufacturer's instructions (reference L4610).

The proteins incorporate S³⁵-methionine (Amersham Pharmacia).

The products obtained in vitro are analyzed by SDS-PAGE electrophoresis.

After electrophoresis, the gel is placed in a fixing buffer (5% ofmethanol, 15% of acetic acid, 80% of water) for half an hour and thesignal is amplified by immersing the gel in the Amplify product fromAmersham Pharmacia (Ref: NAMP100).

A Kodak Biomax MR film is then exposed on the dried gel for a periodranging from one hour to one week, and then developed.

Example 8 Protocol for the Coprecipitation of the Proteins

30 μl of the sepharose-glutathione beads coupled to the GST fusionproteins, after standardization of the amounts, are rinsed in buffer B(1% NP40, 50 mM Tris-HCl, 150 mM NaCl, 2 μl/ml leupeptin, 1% aprotinin,1 mM ABESF).

5 to 10 μl of the in vitro translation product as obtained in Example 3are then added, depending on the amount of the product observed byautoradiography.

After contact overnight, the beads are rinsed 10 times with buffer A NP40, without antiproteases.

Analysis is carried out by SDS-PAGE and autoradiography.

It is thus possible to show the binding between the TSIP4 and TSIP2proteins.

Example 9 Protocol for Screening Compounds Which Interfere with theTSIP4-TSIP2 Binding

The assays described in Examples 5 to 8 are carried out, adding thecompounds which it is desired study to the step of Example 8, andcomparison is made with the results obtained when the compounds are notadded.

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